Objective

The objectives of this concept are to 1) generate new or improved models for the study of amelogenesis that accurately reflect the developmental stage or physiological process they are intended to represent, and 2) validate those models to ensure they are robust and reproducible. These efforts may include establishing cells in the ameloblast lineage that retain their properties in culture or 3-dimensional co-cultures of various cell types that incorporate multiple biological signals necessary for the differentiation, growth, and function of ameloblast cells. Additionally, the scope of this concept will include an evaluation of existing cell lines and models by diverse research groups to determine their best utility. As such, tools or approaches to validate and study these models will be needed. Long term, it is expected that this effort will bring together scientists in multiple disciplines to accelerate understanding of multiscale processes of enamel formation and will inform strategies to mitigate diseases affecting enamel and associated structures, and enamel restoration.

Background

Enamel formation occurs through the process of amelogenesis, during which ameloblast cells form and secrete the extracellular matrix which eventually matures into the outer hydroxyapatite layer of the tooth. Any perturbation in this complex process from aberrant amounts of unresorbed protein in the extracellular enamel matrix, to inadequate calcium and ion transport, can lead to hypomineralized or malformed enamel. These imperfections in enamel subject the whole tooth to wear or caries attack, typically leading to subsequent failure of the tooth and decreased quality of life. In genetic conditions such as amelogenesis imperfecta (AI) in which certain enamel proteins are mutated, the teeth of these children are weak and often require repeated and increasingly progressive restorations to regain partial function. AI can manifest clinically in many forms from hypoplastic to hypomineralized enamel, and not only requires substantial clinical care, but also has psychosocial sequelae affecting the patient. AI is a challenge to study because it is a genetically and clinically diverse group of conditions. Another hypomineralized enamel condition, molar-incisor hypomineralization (MIH) which affects a specific subset of teeth, has a global prevalence of 14% among children under 10 years of age. The latest report scouring 70 studies across the world accentuates the lack of information on MIH in the United States. Its etiology is currently unknown but associations have been made with exposure to chemicals in the environment, and studies are needed to understand and prevent MIH in the US. Both newly recognized and long-established diseases affecting enamel manifest themselves early in development and are mostly irreversible after teeth have been formed and erupted.

Over the past decades, models of these enamel disorders have been generated to study different aspects of the pathways involved. Genetic manipulation and proteomic tools have revealed many of the major players in amelogenesis, and both animal models and cell lines have proven useful. The rodent with its continuously erupting incisor has been a model organism for the study of the continuum of amelogenesis where all the enamel formation stages are visible at once. Although studies in- and with cells derived from- these models have produced a wealth of important basic science knowledge, given the lack of continuous tooth eruption in humans, direct translatability of such models to human applications is likely to be limited. This barrier is gradually being overcome with genomic techniques in which animal models, such as the mouse, can be sapienized. Additionally, larger animal models such as the pig could advance translation of in vivo investigations especially if there is a closer semblance to human physiology. Nevertheless, additional new animal models are needed to expand this repertoire. Currently, the research community most commonly uses mouse and rat ameloblast-like cell lines representing secretory and maturation stages, and there is ongoing work to develop those derived from induced pluripotent stem cells (iPSC). Despite the availability of some cell lines, little direct comparison has been made to analyze the utility of these cells. Different conditions and handling may be contributing to mixed results seen in various laboratories as the highly orchestrated process of amelogenesis requires multiple signals and cues from the surrounding microenvironment and other cells. The next frontier lies in charting the appropriate animal models and the proper conditions and factors that are necessary to recapitulate amelogenesis reliably and reproducibly in ex vivo and in vitro models, for accelerating research in enamel formation and restoration.

Gaps and Opportunities

Though the major protein players have been identified and the high-resolution structure of enamel has been obtained, the process of its formation remains difficult to piece together because of its complexity, and is compounded by results from different laboratories that do not always reconcile well with each other. Multiple attempts to engineer artificial enamel have failed to reproduce the macromolecular interweaving structure of enamel rods or recapitulate the unique mechanical properties of natural enamel. A static in vitro method involving organic and inorganic molecular components lacks the dynamic variables that cells provide.

In order to produce enamel biomimetically, knowledge of the detailed mechanisms of amelogenesis are needed and would require cell lines with reproducible, predictable and physiologically-relevant properties. Addressing the lack of appropriate cell lines from presecretory to maturation stages would allow examination of essential molecular processes- such as ion and mineral transport into the enamel space, protein secretion and removal, crystal nucleation and mineralization, and influences from extrinsic factors such as fluoride, bisphenol A, and other environmental exposures- which may perturb amelogenesis. Consistent and well-defined models would allow for controlled manipulation of environmental variables and provide insight into how those variables affect enamel crystal formation directly or indirectly through stresses on the ameloblast cells. More importantly, these models need to be more physiologically relevant to humans so that findings in the laboratory can translate to the clinic.

Recent scientific and technological advances in stem cell biology and multiscale in vitro systems from other disciplines may prove useful to overcome current research gaps in enamel generation and regeneration. For example, organoid and 3-dimensional tissue culture systems incorporating multiple interacting tissue types (also known as Microphysiological Systems or Tissue Chips) are being utilized to mimic complex tissues and organs. Incorporating ameloblasts into such bioengineered in vitro platforms could be highly beneficial, given their polarized nature, requirement for positional cues, and high metabolic rates. Studies of single ameloblasts in culture would need to be analyzed alongside cells in organ culture to determine differences in cell movement, behavior, morphology, and function. An overwhelming amount of evidence indicates that 3-dimensional co-cultures emulate best the native environment by providing biological and mechanical cues that are not present in simpler culture systems and represent an open area for adaptation in enamel research.

Additional opportunities are also available in the field of cellular reprogramming and controlled differentiation of iPSC towards the ameloblast lineage. While there are advances in autologous transplantation of whole tooth germs and generation of tooth-like structures from iPSCs for regenerative medicine purposes, basic science studies are still needed to dissect the physiological processes and regulation within these cells. Gene editing techniques such as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) are allowing unprecedented ease of manipulating highly specific genetic targets. Not only is CRISPR-Cas9 useful for correcting genetic abnormalities in patient-derived iPSCs, it can also be used to inactivate or insert mutations to create cell lines and animal models. The ability to knockin/out multiple genes simultaneously has the potential to recapitulate complex diseases and conditions, ultimately contributing to our understanding of the cellular and molecular processes in health and when they go awry in disease.

NIH values the need for high-quality resources to conduct research as are many investigators who depend upon these biological resources such as cell lines, ex vivo, and animal models. In order to support the effort on NIH’s policy on Rigor and Reproducibility, there is a need to ensure that the cell lines and animal models which are widely used by researchers in the enamel field are validated and robust. To this end, NIDCR will support projects to generate new physiologically-relevant models at different stages of amelogenesis as well as the validation of these new and any existing models and cell lines.

The scope of this concept includes but is not limited to:

Development

Generating ameloblast cells that can prove useful in mechanistic studies from pre-secretory to maturation stages

Determining chemical, biological, and mechanical variables that can reliably support and maintain undifferentiated pre-ameloblast cells, as well as for controlled differentiation into various ameloblast stages

Engineering iPSC that can reproduce the developmental stages of ameloblasts

Developing chambers, devices, scaffolds, and the like for recapitulation of the ameloblast’s native environment

Developing suitable animal models and cells derived from organisms other than mouse and rat

Engineering supporting cells, tissues, or other extracellular structures necessary for the proper growth, development, and function of ameloblasts

Validation

Validating existing and newly generated cell lines to ensure they reproduce the structure and function of ameloblast cells across stages of differentiation and maturation

Establishing consensus standards and best practices for generating these models

Standardizing criteria for evaluating these models

Fostering collaboration among different groups to test the utility of these models

Involving different laboratories to ascertain the robustness and sustainability of these models

Creating open access databases to facilitate storing and sharing of protocols and detailed data on expected behavior and phenotypes of these models

Verifying that different laboratories can reproducibly maintain and study these models

Current Portfolio Overview

Projects focusing on amelogenesis comprise approximately 10% of the NIDCR portfolio. Among all previously funded amelogenesis grants, more than 90% involve the use of animals or cell models.

Individuals and Groups Whose Input was Solicited for This Initiative

A group of fourteen participants were invited to attend an NIDCR-sponsored workshop on this subject. These individuals represented areas of expertise in stem cell biology, ion transport, transgenic animal models, cellular models, transcriptomics, structural biology, and tissue engineering. Their presentations and discussions on the gaps and needs helped provide input to refine this Concept. Public comments specific for this concept were solicited on NIDCR’s website from August 4-September 5, 2017.

Alignment with Institute Goals and Strategic Plan

This initiative is aligned with the NIDCR Strategic Plan 2014-2019, Goals I and II, “Support the best science to improve dental, oral, and craniofacial health” and “Enable precise and personalized oral health care through research” respectively. Specifically, the initiative aligns with objectives I-1, I-2, and II-1 that “Enable basic research to advance knowledge of dental, oral, and craniofacial health”, “Promote development and use of comprehensive, interoperable databases and informatics resources to advance prevention, diagnosis, and treatment of dental, oral, and craniofacial diseases”, and “Support research toward precise classification, prevention, and treatment of dental, oral, and craniofacial health and disease”.